U b i q u i t i n - prote in conjugates

Harris Busch and Ira L.Goldknopf Dept. of Pharmacology, Baylor College of Medicine, Houston, TX 77030, U.S.A.

Summary

The data available at present indicates there are three distinct functions of ubiquitin, two of which are related to protein conjugation. The first of these has been extensively studied by our laboratory and others interested in nucleosomes and changes in chromatin states. The ubiquitin-histone (Ub-2A, Ub-2B) conjuga- tion reaction now appears to be a very dynamic process. In the deconjugation (lyase) reaction, both the histone 2A and the ubiquitin are left intact and in a form which makes possible ready reconjugation. Accordingly, this may be a mechanism for 'moment-to-moment ' control of the genome.

The second function in which ubiquitin is conjugated involves proteolytic activity. This activity is correlated with protein turnover. In this process, the ubiquitin-protein conjugate apparently serves as a 'signal' for the protease cleavage of the protein. The released ubiquitin is also intact and is probably available for reconjugation.

In the third function, ubiquitin was suggested to serve as a 'hormone'. The studies thus far have been carried out primarily on induction of T- and B-lymphocytes, reduction or delay of Coombs' positivity and reduction of spleen weight. The precise physiological role of this reported function is still unclear, particularly because the ubiquitin used was probably not the physiologically active form.

Ubiquitin structure

The small polypeptide, ubiquitin (Ub), has a molecular weight of 8,565 and contains 76 amino acid residues. It was initially purified by G. Goldstein and his associates in the course of studies on peptides of the thymus. With the aid of a radioimmunoassay for this peptide, it became apparent that it. was widely distributed in plant, animal, yeast and bacterial cells (1). The sequence determined for amino acids 1-74 of thymus ubiquitin (2) was:

1 5 10 Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-

15 20 Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-

25 30 Ile-Glu-Asn-Val-Lys-A1a-Lys-Ile-Gln-Asp-Lys-

35 40 Glu-Gly-Ile-Pro-Pro-Asp-Gln-Gln-Arg-Leu-Ile-

45 50 55 Phe-Ala-Gly-Lys-Gln-Leu-Glu-Asp-Gly-Arg-Thr-

60 65 Leu-Ser-Asp-Tyr-Asn-Ile-Gln-Lys-Glu-Ser-Thr-

70 74 75 76 Leu-His-Leu-Val-Leu-Arg-Leu-Arg-Gly-Gly

Their sequence was notable for NH2-terminal methionine (the only one in the molecule) and the arginine at position 74. The sequence of ubiquitin was confirmed by Low and A. Goldstein (3) who independently studied the polypeptides of thymus and referred to this peptide as polypeptide B1. In

Molecuiar and Cellular Biochemistry 40, 173-187 (1981). 0300-8177/81/0403 0173/$03.00. © 1981, Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

174

'active ubiquitin' the C-terminus is Arg-Gly-Gly76 (4-8). Preliminary analysis of the structure of the amino-terminal octapeptide of ubiquitin from celery showed that six of the eight residues were the same as for thymus ubiquitin.

What is most notable about the sequence in relationship to the three-dimensional structure of ubiquitin is that this molecule is extremely resistant to tryptic digestion despite the presence of seven lysine residues and four arginine residues (2). It is likely that this structure is tightly coiled in such a way that the basic amino acids are unavailable to the active sites of trypsin, and other proteolytic enzymes. It was only after maleylation that the molecule was susceptible to the cleavages necessary for structural analysis. Studies with nuclear mag- netic resonance accounted for the resistance to cleavage, i.e., ubiquitin has a highly globular, compact, pH and temperature resistant conforma- tion requiring 7 M guanidine hydrochloride to denature it (9). On the basis of the prediction of protein confromation by Chou and Fasman (10), the NH2-terminal 15 residues (12%) were suggested to be a pleated sheet and the COOH-terminal (28%) was suggested to be a helical region (11). Its activity as a hormone is controversial (12) but this may reflect the absence or presence of the C-terminal Gly-Gly dipeptide. Increased interest in ubiquitin has been stimulated by studies indicating that a number of proteins contain it as all or part of their structures (Table 1).

Ubiquitin-histone conjugates

Ub-2A (protein A24) was first found in the group of 100 0.4 N H~SO4 soluble proteins of nucleoli and nuclei resolved by two-dimensional polyacrylamide gel electrophoresis (13, 14). The presence of ubiquitin in histone was unsuspected and only was found after purification (15) of Ub-2A (protein A24), peptide analysis (16) and amino acid sequencing procedures (17). We were very surprised to find that the protein had a 'Y-shape' with 2 N-terminals and 1 C-terminal amino acid (14).

When the NH2-terminal sequence of Ub-2A (protein A24) was published (17), it was apparent (18) that this was the sequence of ubiquitin (Ub). It was established that ubiquitin was covalently linked to the histone 2A, specifically on the e-amine of lysine 119 (4). This remarkable structure has led to a series of investigations on similar structures in other proteins and on the function of such linkages.

Two-dimensional polyacrylamide gel electro- phoresis (13) (Fig. la) showed that Ub-2A (protein A24) migrated slightly slower than the histones in the urea-acetic acid first dimension and between histone H1 and H2B in the sodium dodecyl sulfate- containing second dimension (19). Ub-2A (protein A24) was in acid extracts of whole nuclei (Fig. lb); its relatively similar amount in nuclear, nucleolar, and extranucleolar extracts indicated that Ub-2A (protein A24) was present throughout the cell nucleus (14).

Table 1. Studies identifying various proteins as free or conjugated ubiquitin.

Form and cellular Original nomenclature Isolation Structural identification localization

Cytoplasmic free UBIP, ubiquitin Goldstein, el al., 1975 (1) a Schlesinger et al., 1975 (2) Peptide of bovine Pars Intermedia Seidah et al., 1978 (83) a Seidah et al., 1978 (83) APFI Ciechanover el al., 1978 (61) b Hershko et al., 1980 (65) Cytoplasmic free

and conjugated Nuclear free

Nuclear conjugated

Ubiquitin H M G20 Nonhistone protein S A24 = H2A + Ub

uH2B = H2B = Ub

Goldknopf et al., 1978 (25) Walker et al., 1978 (84) Marushige & Dixon, 1971 (85) Goldknopfe t aL, 1975 (15)

West & Bonner, 1980 (48)

a Wilkinson et al., 1980 (62) a Golfknopf et al., 1978 (25) a Walker et al., 1978 (84) a Watson et al., 1978 (26) b Goldknopf & Busch, 1975 (16) b Goldknopf & Busch, 1977 (3) a Olson et al.. 1976 (17) d Hunt & Dayhoff, 1977 (18) a'UWest & Bonner, 1980 (48)

a Identified as ubiquitin b Identified as conjugated ubiquitin c Determination of amino terminal sequence d Identity to ubiquitin determined by amino acid sequence comparison

NORMAL RAT LIVER NUCLEOLAR PROTEINS

I I

3

1

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a

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Bt q b

A2~ f BSL

B~3 %

B2

Cb

RAT LIVER NUCLEOLAR PROTEINS

18

c A I

REGENERATING RAT LIVER I I NUCLEO[AR PROTE[ NS

24-25

3

175

b . . . .

Fig. 1. Two-dimensional polyacrylamide gel electrophoresis of 0.4 N H2SO4-soluble proteins. Electrophoresis was from right to left in the first dimension in 10% acrylamide-4 M urea-0.9 acetic acid. The second dimension was from top to bottom in 1% SDS-0.1 M sodium phosphate (pH 7.1) 6 M urea. The histones are spots GAR (histone 4), A 1 (histone 2A), A2 (histone 2B), A3, 4 (histone 3), and A l 1, 17-19, (histone 1 ). Five hund red micrograms of protein were used for each gel. (a) and (d) from Ballal (22), (b) from Yeoman et aL (14); and (c) from Ballal et al. (21). Protein A24 is Ub-2A.

Ub-2A was of physiological interest because its nucleolar content was markedly decreased in rat liver during thioacetamide administration and in regenerating liver after partial hepatectomy (20-22) (Fig. lc & d). Since these nucleoli have high levels of rRNA synthesis (23, 24), the decreased Ub-2A (protein A24) content as well as the presence of free ubiquitin in some active fractions (25-27), sug- gested that cleavage of Ub-2A (protein A24) might be related to increased gene activity.

Isolation of electrophoretically homogeneous Ub- 2A (protein A24)

of Ub-2A (protein A24) (15) was calf thymus chromatin preextracted with 0.35 M NaC1 and 0.5 M perchloric acid (Fig. 2a). Ub-2A (protein A24) was in the 0.4 N H2SO4 extract along with other histones (Fig. 2a).

Ub-2A (protein A24) was fractionated on Sephadex G-100 (Fig. 2b) and then on slab gels (Figs. 3 & 4); it migrated in two-dimensional polyacrylamide gel electrophoresis as a single com- ponent (Fig. 2c).

The primary structure of Ub-2A (protein A24)

Presence of histone 2A in Ub-2A (protein A24)

The starting material employed for purification The amino acid compositions of Ub-2A (protein

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Fig. 3. Purification of protein A24 by preparative electro- ph0resis. (a) An Amid o black-stained vertical side strip cut from a preparative 10% polyacrylamide slab gel after electrophoresis of the pooled fractions obtained from column chromatography on Sephadex G-100 (Fig. 2b). The position of protein A24 and histones 2B (A2) and 3 (A3-5, 7) are indicated. Such vertical strips from both sides and the center were used as a guide to cut out horizontal sections of the unstained remainder of the slabs which contained the protein A24 band. The protein A24 was then obtained by electrophoresis out of the gel sections into dialysis tubing. (b) Reelectrophoresis of purified protein A24 obtained in (a) on 10% polyacrylamide gels (15).

177

A24) and histone 2A (Table 2) (16) showed their similar contents of lysine, histidine, aspartic acid, leucine, and phenylalanine. Ub-2A (protein A24) contained a lower molar ratio of arginine and alanine than histone 2A. The contents of threonine, glutamic acid, and proline were higher in Ub-2A (protein A24), and it had a higher acidic-basic amino acid molar ratio than histone 2A. Neither protein contained tryptophan. Histone 2A contain- ed no methionine. The methionine residue was one amino terminal amino acid of Ub-2A (protein A24) as shown by dansylation.

A comparison of the peptide maps of these proteins revealed striking similarities (16). The tryptic peptides of histone 2A (28) were present in the corresponding pattern of Ub-2A (protein A24). Similar results were obtained with chymotryptic peptides.

Inasmuch as a) Ub-2A (protein A24) had a methionine NH2-terminus and histone 2A contains no methionine and b) Ub-2A (protein A24) had a molecular weight greater than histone 2A and had additional peptides not in histone 2A upon both tryptic and chymotryptic digestion, it seemed that Ub-2A (protein A24) contained the amino acid

Table 2. Comparison of Ub-2A (protein A24) and histone 2A.

Residue Protein A24 a Histone 2A b (mole %) (mole %)

Trp 0.0 0.0 Lys 11.3 10.9 His 2.4 3.1 Arg 7.4 9.3 Asx 7.3 6.2 Thr 6.5 3.9 Ser 4.5 3.1 Glx 12.3 9.3 Pro 5.6 3.9 Gly 9.2 10.9 Ala 9.6 13.4 Val 4.9 6.2 Met 0.3 0.0 lieu 5.8 4.7 Leu 10.9 12.4 Try 1.3 2.3 Phe 0.9 0.8

Lys + His + Arg 21.1 23.3 Glx + Asx 19.6 15.5 Glx + Asx /Lys + His + Arg 0.93 0.67 Dansylatable NHz-terminal Methionine Blocked (acetyl-Ser)

Molecular weight 27 000 14 000

a Goldknopf et al. (1975) b Yeoman et al.

178

Calf Thymus

NaCI-EDTA

Pellet

Pellet Supt: Tris Soluble Proteins, Discard

~ 0.35 M NaCI 3X

Pellet Supt: HMG and LMG Proteins, Discard

pcA 2x

2X

Supt: NaC1-EDTA Soluble Prote ins , Discard

Pel le t Supt: Histone H1, Discard

S u p t : A24 and H i s t o n e s H2A, H2B, H3 and H4 s a v e

S e p h a d e x G-100

A24 + H2B + H3

Preparative PAGE

A24

Fig. 4. Flow sheetforpreparation ofprotein A24.

P e l l e t : DNA + R e s i d u a l P r o t e i n s , Discard

sequence of histone 2A along with an additional amino acid sequence (16).

The terminal amino acid sequences o f Ub-2A (protein A24)

The peptide designated 16 in Ub-2A (protein A24) was not found by the ninhydr in-cadmium or fluorescamine procedures which require a pr imary amino group (Table 3); peptides 16 (4) f rom histone 2A and Ub-2A (protein A24) (Table 3) had identical staining characteristics, amino acid composi t ion, and positive tests for the acetyl group consistent with the structure of the blocked amino terminal tryptic peptide of histone 2A: N-acetyl-Ser-Gly- Arg.

The fact that Ub-2A (protein A24) contained a second NH2-terminus , a methionine, suggested that it had an addit ional (non-histone) sequence. This unblocked sequence was amenable to automat ic Edman degradat ion without prior separation of the two polypeptides.

The sequence of the first 37 NH2-terminal resi- dues (17)

5 10 Met-Gln-I!e-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys_

Table 3. Analysis of tryptic peptide 16 of protein A24 and histone 2A.

Protein A24 Histone 2A

Staining reactions Fluorescamine Ninhydrin Sakaguchi + + Rydon-Smith + +

Amino acid ratios set (= 1.0) 1.00 1.00 Gly 1.09 1.15 Arg 1.12 1.21

Molar yield a 0.42 0.46 Acetyl group b + +

a Nmoles recovered from tryptic peptide maps/nmole of protein digested.

b Detected as acetyl hydrazide.

15 20 Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-

25 30 Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln-Asp-Lys-

35 Glu-Gly-Ile-Pro-

was identical to the first 37 amino acids of ubiquitin (2, 18) as noted above.

The known COOH-terminal amino acid sequence (29) and rate-limiting steps for carboxypeptidase B and A digestions of histone 2A were:

-His-His-Lys-Ala-Lys-Gly-Lys-COOH

A B

The values in Table 4 obtained by equating the nanomoles oflysine to 1.0 after carboxypeptidase B and to 3.0 after carboxypeptidase A digestions were identical for both proteins (17). Ub-2A (protein A24) had the same COOH-terminal sequence as histone 2A. Quantitative hydrazinolysis indicated that lysine was the sole COOH-terminal amino acid of Ub-2A (protein A24), and its molar yield was identical to that of histone 2A (4).

The branched tryptic peptide of Ub-2A (protein A24)

The detection of two amino-termini and one carboxyl-terminus (4) led to the conclusions that the Ub-2A (protein A24) molecule was branched and that the ubiquitin was linked to histone 2A in a manner that prevented detection of its carboxyl- terminus. A search for an altered tryptic peptide of Ub-2A (protein A24) (4) showed tryptic peptide 17' from Ub-2A (protein A24) had a slightly different

179

electrophoretic mobility from peptide 17 of histone 2A (Fig. 5). The amino acid composition and carboxyl terminus of 17' of Ub-2A (protein A24) (Table 5) were the same as that of peptide 17 (residues 119-124) of histone 2A except for the presence of two additional glycine residues.

The first cycle of Edman degradation of the

i!

Fig. 5. Two-dimensional tryptic peptide maps of (a) histone 2A and (b) protein A24. Vertical dimension is chromatography and horizontal dimension is electrophoresis. Spots 1-16 are common to both proteins. Note the positions of peptides 16 which were negative to the ninhydrin-cadmium stain used in the maps. Note also the position of peptides 17 of histone 2A and 17' of protein A24, which differ slightly in electrophoretic mobility.

Table 4. Ratios of free amino acids released from protein A24 and histone 2A digestion with carboxypeptidases A and B.

Step 1 a residues released from Step 2 b residues released from Amino acid

Protein A24 Histone 2A Protein A24 Histone 2A

Lysine 1.0 1.0 3.0 3.0 Histidine 0.1 0.1 0.9 1.0 Alanine 0.1 0.1 0.9 0.7 Glycine 0.1 0.1 0.9 0.6

a Carboxypeptidase B: 60-min digestion followed by boiling for 5 min. b Carboxypeptidase A: After step 1, 60-min digestion.

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Table 5. Amino acid ratios and carboxyl-termini of peptides 17 of histone 2A and 17' of protein A24.

17 17'

Gly - 1.64 Lys 1.67 1.68 His 1.71 1.61 Thr 0.91 0.93 Ser (= 1.00) 1.00 1.00 Glu 0.95 0.94 COOH-terminus a Lys (0.57) b Lys (0.64) e Molar yield c 0.67 0.74

a Data obtained by hydrazinolysis, only lysine was found. b Data in parentheses are molar yield of carboxyl-terminal

lysine. c Nmoles of peptide recovered from tryptic peptide maps/nmole

of protein digested.

Ub-2A (protein A24) peptide was consistent with a branched structure arising f rom the addit ional two glycines being at tached to the rest of the peptide in an isopeptide linkage to the e-amino group oflysine (cf below). Coupling with phenylisothiocyanate occurred at the c~-NH 2 groups of both lysine and glycine (4). Cyclization produced two thiazolinones, one containing glycine and one containing lysine with a glycine residue at tached to its e-NH2 group. HI hydrolysis of these thiazolinones released two glycine residues and one lysine residue. In addition, conversion of the thiazolinones to phenyl thiohy- dantions (PTH) produced sufficient PT H glycine to account for half of the glycine in the thiazolinones.

Table 6. Sequential Edman degradation of peptides 17 and 17 a.

Edman cycle

Amino acid 1 2 3 4

Peptide 17 of histone 2A Glys Lys Thr Glu Ser

Peptide 17' of protein A24 Gly Lys Thr Glu Ser

0.5 1.0

0.9

1.6 0.5 0.4 0.1

1.1 0.8

0.3

0.3

a Nmoles of amino acids/nmoles of peptide released from thiazolinones after hydrolysis with HI.

Gas chromatographic analysis of phenyl th iohydan- tions showed there was 0.9 nmole of PTH-glycine/ nmole of peptide, or approximate ly one-half of the amoun t obtained f rom hydrolysis of thiazolinones. These data and subsequent cycles of Edman degra- dat ion (4), which produced essentially identical results for both peptides (Table 6), supported the branched structure of this peptide and provided the first evidence for the Gly-Gly bond (Fig. 6).

Trypsin t reatment of Ub-2A (protein A24) yielded intact ubiquitin (4) as expected f rom the known resistance of ubiquit in to trypsin (2). The Arg-Gly-Gly linkage was cleaved under these con- ditions (4). Pret reatment of Ub-2A (protein A24) with the arginine blocking reagent 1,2-cyclohexa- nedione (30), followed by trypsinization produced (5) a trypsin-resistant polypeptide which included the branched peptide 17' linked to arginine 74 of the carboxyl- terminus of ubiquitin and the glycines of peptide 17'. Recently two-dimensional polyacryl- amide gel electrophoretic mobility and amino acid composi t ions of fragments of Ub-2A (protein A24) f rom cleavage at tyrosines with N-bromosuccini- mide accounted for the whole structure of Ub-2A (protein A24) (6) as shown in Fig. 7.

The novel bifunctional structure of Ub-2A (pro- tein A24) immediateley raised questions about other bifunctional proteins. Other proteins contain two functional units; /3-1ipotropin has both

A. Histone 2A peptide 17:

L y s - T h r - G l u - S e r - ( H i s ) 2 - L y s

Edman cycles: 1 2 3 4 comp.hydraz.

Residue no. in Histo~e 2A: 119 120 121 122 123-4 125

B. Protein A24 peptide 17':

O H O . 4 II

H2N-CH2-C-N-CH2-C-NH

(Gly) (Gly) ]

(~H2) 4 1 P

Edman c y c l e s : H2N-CH- 6/-Thr-Glu-Ser - (His) 2 -Lys (Lys) 1 2 3 4 comp. hydraz .

Fig. 6. Structure and outline of proof of structures of peptides (A) 17 and (B) 17'. Note the identical sequence Lys-Thr-Glu-Ser- His-His-Lys in both peptides and the position number of these amino acid residues in the histone 2A sequence. Comp., amino acid composition, Hydraz., hydrazinolysis.

181

Ubiquitin Portion

1 5 i0 H2N-Met--Gln-lle-Phe-Val-Lys-Thr-Leu-Thr-Gly-

ii 15 20 Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-

21 25 30 Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-

31 35 40 Gln-Asp-Lys-Glu-Gly-lle-Pro-Pro-Asp-Gln-

41 45 50 Gln-Arg-Leu-Ile-Phe-Ala-Gly-Lys-Gln-Leu-

51 55 60 G1u-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-Asn-

61 65 70 Ile-Gln-Lys-Glu-Ser-Thr-Leu-His-Leu-Val-

0

Histone 2A Portion

1 5 i0 acetylSer-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-Arg-Ala-

15 20 25 Lys-Ala-Lys-Thr-Arg-Ser-Ser-Arg-Ala-Gly-Leu-Gln-Phe-

30 35 Pro-Val-Gly-Arg-Val-His-Arg-Leu-Leu-Arg-Lys-Gly-Asn-

40 45 50 Tyr-Ala-Glu-Arg-Val-Gly-Ala-Gly-gla-Pro-Val-Tyr-Leu Ala-

55 60 65 A l a - V a l - L e u - G 1 u - T y r - L e u - T h r - A l a - G l u - I l e - L e u - G l u - L e u -

70 75 Ala-Gly-Asn-Ala-Ala-Arg-Asp-Asn-Lys-Lys-Thr-Arg-lle-lle-

80 85 90 Pro-Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp-Glu-G1u-

75 I00 105 Leu-Asn-Lys-Leu-Leu-Gly-Lys-Va1-Thr-I1e-A1a-Gln-Oly-

71 11 Leu-grg-Leu-Arg-Gly-Gly - C ii0 115

( ~ . ~ . ~ Gly-Val L e u - P r o - A s n - I l e - G l n - A l a - V a l - L e u - L e u - P r o - L y s - N H

a 120 125 129 Lys-Thr -Glu-Ser-His-His-Lys-AI a-Lys-Gl y-Lys-COOH •

Fig. 7. The complete amino acid sequence of Ub-2A (protein A24) wherein the carboxyl terminal glycine 76 of ubiquitin is attached to the e-NH 2 of lysine 119 of histone 2A by an isopeptide linkage.

melanocyte-stimulating hormone and/3-endorphin (31-34), but both units are within a single polypep- tide chain. The linkage of the histone 2A and ubiquitin by an isopeptide suggested a post-trans- lational conjugation. Isopeptide linkages have been found in collagen (35), fibrin (36, 37), peptidogly- cans (38), wheat germ (39), bovine colostrum (40), hair medulla protein (41), chorion of rainbow trout (42), and E. coli ribosomal protein S l l (43). However, histones and nonhistone chromosomal proteins had not been shown to contain such a linkage previously and the ubiquitin-histone 2A bond was the first such conjugate demonstrated.

In most systems studied, approximately 10% of histone 2A is in the form of protein A24 (15, 44, 45) and this quantitative distribution is the same for all the postsynthetic modification (46, 47) and amino acid sequence (44, 45) variants of histone 2A.

Recently, a ubiquitin adduct of histone 2B, Ub-2B, has been described (48) in which 1-1.5% of histone 2B is conjugated in the carboxyl terminal portion with ubiquitin (48). This concentration of Ub-2B in the nucleus is approximately 1/10 that of Ub-2A (protein A24). Both sequence variants of histone 2B are conjugated to the same extent.

C h r o m o s o m a l local izat ion - the presence of Ub-2A (protein A24) in nucleosomes

The distribution of Ub-2A (protein A24) in chromatin (15) is illustrated in Figs. 8 and 9. Ub-2A (protein A24) and the histones were not detected in the saline-EDTA (0.075 M NaC1-0.025 M EDTA, pH 8.0) (Fig. 8b) or the Tris (Fig. 8c) washes, but were found in the 0.4 N H2SOn-soluble proteins of chromatin (Fig. 8d). Ub-2A (protein A24) and the histones were not solubilized when the chromatin was treated with 0.35 M NaC1 (Fig. 9a) (15), which extracted the high mobility group (HMG) and low mobility group (LMG) nonhistone chromosomal proteins (49).

The 0.4 N H2SOa-soluble proteins of the chro- matin residue contained Ub-2A (protein A24) and the histones (Fig. 9b). The difference in the solubil- ity of Ub-2A (protein A24) and histone H1 (Al l , 17-19) was evident after the extraction from chro- matin of histone H 1 with 0.6 M NaCI or with 0.5 M HC104. Under these conditions, Ub-2A (protein A24) and histones 2A, 2B, 3 and 4 were not extracted (Fig. 2a & 9d, respectively). Ub-2A (protein A24) accounted for 1.9% of the total of histones 1, 2A, 2B, 3 and 4.

182

a

Fig. 8. Two-dimensional polyacrylamide gel electrophoretic analyses of the distribution of rat liver nuclear proteins during chromatin preparation. (a) 0.4 N H2SO 4 soluble nuclear proteins; (b) proteins solubilized in two washes of 0.075 M NaC1, 0.025 M EDTA, pH 8.0, 1 mM PhCH2SO2F; (c) proteins solubilized in two washes of 0.01 M Tris, pH 8.0, I mM PhCH2SO2F; (d) 0.4 N HzSO4-soluble chromatin proteins.

When the histones and most nonhistone chromo- somal proteins were extracted from chromatin with 3 M NaCI-7 M urea, Ub-2A (protein A24) was extracted (Fig. 9c). Ub-2A (protein A24) and the histones reassociated with the DNA by one-step or gradient analysis to low ionic strength. Ub-2A (protein A24) had similar binding characteristics to histones 2A, 2B, 3 and 4, although it was present in much lower amounts than histones.

Thus, Ub-2A (protein A24) is solubilized f rom chromatin along with histones 2A, 2B, 3 and 4 (15). Two each of those histones compose the octameric globular nucleosome protein core particle (50). Ub-2A (protein A24) is found in a subset of nucleosomes where it replaces free histone 2A in the core octamers (51, 52, 44). The interactions between

histone 2A and other core histones (50, 53, 54) and histone 1 (55-57) are apparently not disrupted by ubiquitination (44, 52, 55). It has been speculated that the presence of Ub-2A (protein A24) might affect the flexibility of adjacent nucleosomes (58, 59) inhibiting condensation of the chromatin (58-60). Interestingly Ub-2B has also been found in nucleosome cores (48). Apparently, free ubiquitin is not a DNA binding protein (86, 87, 1 l), al though it has been found among the high mobility group proteins (84).

Intracellular proteases and ubiquitin protein conjugates

In early studies on factors involved in intra-

183

a

C ~ A T I N 025M NaCI SOLUBLE PROTEINS

CHROMAT~N 3~M N~CI. 7M UREA SOLUBLE PROTEINS

POST 0,35M NaCI DNP 0.4N SOLUBLE ~ E I N S

b d

Fig, 9. Distribution of rat liver chromatin proteins during extraction with various solutions. Electrophoresis conditions were the same as in Figs. 1 and 2. (a) Proteins solubilized from chromatin by three extractions of 0.35 M NaC1, 0.01 M Tris, pH 8.0, 1 mM PhCH2SO2F; (b) 0.4 N H2SO4-soluble proteins of the residual deoxyribonucleoprotein after 0.25 M NaCI extractions. Note the presence of histone I (A 11, 1.7-19). (c) Proteins dissociated from chromatin in 3 M NaC1, 7 M urea, 0.05 M sodium acetate, pH 6.0, I mM PhCH2SO4F after the DNA was pelleted by centrifugation at 214 000 g for 24 h; (d) 0.4 N H2SO4-soluble proteins of the residual DNP after three (10 ml/g of nuclei) extractions of chromatin with 0.6 M NaCI, 0.01 M Tris, pH 8.0, 1 mM PhCH2SO2F. Note the markedly reduced amounts of histone 1 (spots Al 1, 17-19) compared to (b).

cellular protease activities, Ciechanover et al. (61) found that degrad~/tion of globin was stimulated by ATP and that two polypeptides were involved, i.e., a protease and APF-I which was later shown to be ubiquitin (62). The reaction was dependent upon the concentration of ubiquitin.

Ubiquitin conjugase

Later Ciechanover et al. (63 66) found that ubiquitin formed conjugates by incubation with a reticulocyte fraction retained on DEAE-cellulose.

The system required ATP and Mg ++ and was inhibited by N-ethylmaleimide suggesting an in- termediary-SH linkage existed as with coenzyme A. The conjugates formed were stable on SDS-poly- acrylamide gels and on Sephadex G-75. They resisted acid, alkali, heat, and reduction which led to the conclusion that they were covalently-linked structures.

The proteolysis model showed that there are multiple ubiquitin conjugates of the protein sub- strates used, i.e. lysozymes, globin and lactalbumin. Like the ubiquitin linkage described for Ub-2A

184

(protein A24), these linkages are also isopeptides. Unlike these lifikages, the linkage in Ub-2A is very specific, i.e., only one special site is conjugated.

The conjugase required ATP; other nucleoside triphosphates were much less effective. The enzyme that carries out the activation reaction exchanges pyrophosphate with ATP. The linkage was acid stable but was labile to alkali, hydroxylamine, borohydride and mercuric salts (66). Accordingly an SH group ofconjugase is essential for formation of the ester. The essence of the reaction (67) is:

o II

Ub-GlyCOOH + ATPF"qUb-Gly-C-OAMP + PPi 76 76

O O

II Ub-GIy-C-OAMP ÷ HS-Eq ~Ub-GIy-C-S-E + AMP

76 76

This activated ubiquitin then is transferred with the aid of other factors (68) to multiple e-NH 2 groups of lysines of proteins prior to degradation.

C-terminal-Gly-Gly in active ubiquitin

Wilkinson et al. (62) confirmed that ubiquitin formed the same conjugates in the reticulocyte system as were noted above. Inasmuch as there was some uncertainty about the precise amino acid of the Arg-Gly-Gly ubiquitin terminus in the forma- tion of the isopeptide linkages, Hershko et al. (67) attempted to identify the ATP-activated terminal amino acid which they found to be glycine.

The problem involved emerged from the sequence studies on calf thymus ubiquitin which has been found by Schlesinger et al. (2) and Low and A. Goldstein (3) to have a C-terminal-Arg-, However, in the studies from our laboratory (4-6), the linkage of ubiquitin to histone 2A was Arg-Gly-Gly. The question then was whether the Gly-Gly was added later or was an intermediate. In this connection, the studies of Watson et al. (26) indicated that in trout nuclear ubiquitin, some termini were Gly, and some Arg- as shown by hydrazinolysis. Hershko et al. (67) treated their ubiquitin-conjugase complexes with NaB3H4, hydrolyzed the product with acid and released ethanolamine derived from the active acid residue. Thus, they concluded that the carboxyl terminus of the activated ubiquitin was glycine. The active form of ubiquitin in protein conjugation probably has an Arg-Gly-Gly carboxyl-terminus

(4-8). Presumably, the loss of glycines in calf thymus ubiquitin resulted from partial proteolysis by a protease of uncertain specificity, although this has not yet been demonstrated. Whether this is a physiological phenomenon or specific processing is uncertain.

Deconjugat ion o f ubiquitin

The recently discovered lyase (69) which cleaves the Ub-2A (protein A24) to histone 2A and ubi- quitin provides a ubiquitin which has two C- terminal glycine residues (7). The activity of this enzyme may be important in the turnover of ubiquitin which has recently become a topic of increased interest (see below).

Functions of ubiquitin conjugates of protein

In the proteolysis system, one can visualize a role for the ubiquitin conjugate as a recognition site for the protease (65). Presumably ubiquitin, which is remarkably insensitive to proteases (2) constitutes either an allosteric binding site or a portion of the protein configuration necessary for the reaction site on the enzyme surface. However, there may be other, more complex possibilities. Exactly how cells identify proteins they no longer need or want and the precise chemistry of the conjugation reac- tions with respect to substrate site and the enzymes involved remain to be elucidated.

It is clear that these reactions are different from those involved in the nucleosomes where the ubi- quitin of Ub-2A (protein A24) turns over rapidly and at different times from the synthesis of the histones (70-71). Accordingly, it is likely that the eukaryotic cells have adapted a mechanism that may be involved in protein turnover to a use in chromatin packing, structure or function that does not involve proteolysis.

The role of ubiquitin in the chromatin is much less clear. One problem that existed from the beginning of our studies was the fact that Ub-2A (protein A24) was decreased in nucleoli of cells stimulated for cell division (regenerating liver) or for hypertrophy of the nucleoli (thioacetamide treatment) (20-22). These results which were clear- cut and readily reproducible were initially thought to relate to the phenomenon of increased rDNA

transcription. In the original Miller spreads (23), no nucleosomes were present along the rDNA seg- ments which were being transcribed (88). The RNA polymerase I had supplanted the nucleosomal structures.

The idea that Ub-2A (protein A24) was decreased on active ribosomal genes has been confirmed recently by Levinger et al. (72) who showed that they are deficient in Ub-2A (protein A24). In this case, the decrease of the ubiquitin-conjugates makes sense in relation to the displacement of nucleo- somes from the rDNA.

A decrease in Ub-2A (protein A24) was also found during repression of gene activity. Matsui et

al. (60) reported that metaphase chromosomes which are essentially collapsed chromatin, con- tained no Ub-2A (protein A24) conjugates and hence, ubiquitin residues were lo~t. In addition, the relatively inactive mature chicken erythrocytes did not contain Ub-2A (protein A24) in significant amounts but in the related transcriptionally active 'reticulocytes' the Ub-2A (protein A24) adduct was present in higher concentration (73). Accordingly, the dichotomy existed that in the transcriptionally active chromatin, Ub-2A (protein A24) was present but that in the active states of the rDNA genes, this conjugate was absent.

The question then was whether Ub-2A (protein A24) was present along active genes. Levinger et al.

(72) showed that active gene regions other than rDNA contained Ub-2A (protein A24) and so did regions containing repetitive DNA. Accordingly, the Ub-2A (protein A24) complex would appear to be distributed rather broadly throughout the chro- matin and may or may not be uniformly spaced in nucleosomes in extended chromatin. Barring the development of new evidence which would point to a more specific role for the Ub-2A (protein A24) adducts (and presumably for its other Ub-nucleo- some adducts), it would seem that this conjugation would serve a structural role, possibly maintaining the chromatin in a state where it would be available for transcriptional reactions (59, 73) rather than the state of marked condensation as in active chro- matin.

Several lines of evidence indicate that the metab- olism of the ubiquitin portion of Ub-2A (protein

185

A24) is a function of nuclear activity. The ubiquitin portion of the molecule turns over throughout the interphase (70, 71). The rates of ubiquitin synthesis and conjugation into Ub-2A (protein A24) are parallel and drop to minimal levels during mitosis (70) when the Ub-2A (protein A24) is absent from metaphase chromosomes (60). Similarly, Ub-2A (protein A24) and free ubiquitin are lost from nuclei during chicken erythropoiesis (73) when transcrip- tion stops (74). Since the mechanisms of conjuga- tion and deconjugation are specific and in progress throughout the cell cycle and in nondividing cells, one question is whether Ub-conjugation is a ran- dom or specific event and as noted above, it is still too early to draw conclusions about this point.

An interesting recent finding (75) is that the cleavage of Ub-2A itself is tied to its formation. In the chicken reticulocyte, the cleavage of Ub-2A, measured as endogenous lyase activity is brisk, but in the mature, inactive erythrocyte, the lyase activ- ity ceases while little Ub-2A (protein A24) is detected (73). Presumably the dynamic equilibrium between free ubiquitin and Ub-2A must be under rigid control.

Marunouchi and his associates (76-78) have utilized an interesting mouse ts mutant which is defective in H1 histone phosphorylation. Under nonpremissive conditions (39 °) they found that Ub-2A (protein A24) disappears from the chro- matin. Ub-2A (protein A24) reappeared in the shift- down to the permissive temperature regardless of whether cycloheximide is present or not. This result suggests that Ub-2A (protein A24) lyase was un- affected at 39 ° but that the conjugase was inacti- vated. They suggest that there is a relationship between the presence of Ub-2A (protein A24) and histone kinase activity, but the precise interaction is not clear (76-78).

Acknowledgement

This research was supported by the Research Program Project Grant CA-10893, P l l , awarded by the National Cancer Institute, the Davidson Fund and the Bristol-Myers Foundation.

186

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Received April 22, 1981.